In this study, it was aimed to reveal the in vitro interactions of combinations of colistin with meropenem, rifampicin and tigecycline in colistin-resistant, biofilm-forming Klebsiella pneumonia. A total of 30 isolates, 15 of which formed biofilms and 15 did not form biofilms, were randomly selected from K. pneumoniae isolates growing in blood samples. The synergy rates of colistin-meropenem, colistin-tigecycline, colistin-rifampicin combinations in planktonic/sessile bacteria are; It was determined as 83,3%/73,3%, 66,6%/33,3%, 100%/60% respectively. Biofilm inhibitory concentration (BIC) of colistin, meropenem, tigecycline, and rifampicin significantly increased after biofilm formation. The synergistic activity seen in the sessile form was independent of the planktonic form. Although a high synergistic effect was observed in the meropenem-colistin combination on sessile bacteria, colistin had very high BIC ​​in all combinations. Large-scale studies are needed in which the number of isolates studied is large, bacterial resistance profiles are evaluated genomically, and various antimicrobial groups are included.

Combining commercial antibiotics with adjuvants to lower their minimum inhibitory concentration (MIC) is vital in combating antimicrobial resistance. Evaluating the ecotoxicity of such compounds is crucial due to environmental and health risks. Here, eugenol was assessed as an adjuvant for 7 commercial antibiotics against 14 pathogenic bacteria in vitro, also examining its acute ecotoxicity on various soil and water organisms (microbiota, Vibrio fischeri, Daphnia magna, Eisenia foetida, and Allium cepa). Using microdilution methods, checkerboard assays, and kinetic studies, the MICs for eugenol were determined together with the nature of its combinations with antibiotics against bacteria, some unexposed to eugenol previously. The lethal dose for the non-target organisms was also determined, as well as the Average Well Color Development and the Community-Level Physiological Profiling for soil and water microbiota. Our findings indicate that eugenol significantly reduces MICs by 75 to 98%, which means that it could be a potent adjuvant. Ecotoxicological assessments showed eugenol to be less harmful to water and soil microbiota compared to studied antibiotics. While Vibrio fischeri and Daphnia magna were susceptible, Allium cepa and Eisenia foetida were minimally affected. Given that only 0.1% of eugenol is excreted by humans without metabolism, its environmental risk when used with antibiotics appears minimal.

Acinetobacter baumannii, which is predominantly responsible for hospital-acquired infections, presents a tremendous clinical challenge due to its increasing antibiotic resistance to colistin (COL), a last-line antibiotic. As a result, the combination of antimicrobial and non-antimicrobial agents is emerging as a more popular treatment approach against infections caused by COL-resistant A. baumannii. This study administered COL and verapamil (VER), that is an antihypertensive and antiarrhythmic agent. We found that the susceptibility of A. baumannii to COL was restored both in vitro and in vivo. Scanning electron microscope and Crystal violet staining showed inhibition of the VER/COL combination on bacterial biofilm formation. Cytotoxicity assay and haemolysis test were used to confirm in vitro safety evaluation. Further experiments using propidium iodide staining revealed that the VER/COL combination improved the therapeutic efficacy of COL by modifying the permeability of bacterial membranes. As demonstrated by reactive oxygen species experiments, the drug combination caused the accumulation of bacterial reactive oxygen species and their eventual death. Additionally, VER/COL treatment significantly reduced the efflux of Rhodamine 123 (Rh123). For the first time, this study identifies the anti-hypertensive drug VER as a COL potentiator against A. baumannii, providing a potential treatment approach against A. baumannii infections and improving patient outcomes.

OBJECTIVE: Ceftriaxone-based antimicrobial therapies for gonorrhea are threatened by waning ceftriaxone susceptibility levels and the global dissemination of the high-level ceftriaxone-resistant gonococcal FC428 clone. Combination therapy can be an effective strategy to restrain the development of ceftriaxone resistance, and for that purpose, it is important to find an alternative antimicrobial to replace azithromycin, which has recently been removed in some countries from the recommended ceftriaxone plus azithromycin dual-antimicrobial therapy. Ideally, the second antimicrobial should display synergistic activity with ceftriaxone. We hypothesized that bacitracin might display synergistic activity with ceftriaxone because of their distinct mechanisms targeting bacterial cell wall synthesis. In this study, we showed that bacitracin indeed displays synergistic activity with ceftriaxone against Neisseria gonorrhoeae. Importantly, strains associated with the FC428 clone appeared to be particularly susceptible to the bacitracin plus ceftriaxone combination, which might therefore be an interesting dual therapy for further in vivo testing.

Cold atmospheric-pressure plasma (CAP) has emerged as a potential alternative or adjuvant to conventional antibiotics for the treatment of bacterial infections, including those caused by antibiotic-resistant pathogens. The potential of sub-lethal CAP exposures to synergise conventional antimicrobials for the eradication of Pseudomonas aeruginosa biofilms is investigated in this study. The efficacy of antimicrobials following or in the absence of sub-lethal CAP pre-treatment in P. aeruginosa biofilms was assessed. CAP pre-treatment resulted in an increase in both planktonic and biofilm antimicrobial sensitivity for all three strains tested (PAO1, PA14, and PA10548), with both minimum inhibitory concentrations (MICs) and minimum biofilm eradication concentrations (MBECs) of individual antimicrobials, being significantly reduced following CAP pre-treatment of the biofilm (512-fold reduction with ciprofloxacin/gentamicin; and a 256-fold reduction with tobramycin). At all concentrations of antimicrobial used, the combination of sub-lethal CAP exposure and antimicrobials was effective at increasing time-to-peak metabolism, as measured by isothermal microcalorimetry, again indicating enhanced susceptibility. CAP is known to damage bacterial cell membranes and DNA by causing oxidative stress through the in situ generation of reactive oxygen and nitrogen species (RONS). While the exact mechanism is not clear, oxidative stress on outer membrane proteins is thought to damage/perturb cell membranes, confirmed by ATP and LDH leakage, allowing antimicrobials to penetrate the bacterial cell more effectively, thus increasing bacterial susceptibility. Transcriptomic analysis, reveals that cold-plasma mediated oxidative stress caused upregulation of P. aeruginosa superoxide dismutase, cbb3 oxidases, catalases, and peroxidases, and upregulation in denitrification genes, suggesting that P. aeruginosa uses these enzymes to degrade RONS and mitigate the effects of cold plasma mediated oxidative stress. CAP treatment also led to an increased production of the signalling molecule ppGpp in P. aeruginosa, indicative of a stringent response being established. Although we did not directly measure persister cell formation, this stringent response may potentially be associated with the formation of persister cells in biofilm cultures. The production of ppGpp and polyphosphate may be associated with protein synthesis inhibition and increase efflux pump activity, factors which can result in antimicrobial tolerance. The transcriptomic analysis also showed that by 6 h post-treatment, there was downregulation in ribosome modulation factor, which is involved in the formation of persister cells, suggesting that the cells had begun to resuscitate/recover. In addition, CAP treatment at 4 h post-exposure caused downregulation of the virulence factors pyoverdine and pyocyanin; by 6 h post-exposure, virulence factor production was increasing. Transcriptomic analysis provides valuable insights into the mechanisms by which P. aeruginosa biofilms exhibits enhanced susceptibility to antimicrobials. Overall, these findings suggest, for the first time, that short CAP sub-lethal pre-treatment can be an effective strategy for enhancing the susceptibility of P. aeruginosa biofilms to antimicrobials and provides important mechanistic insights into cold plasma-antimicrobial synergy. Transcriptomic analysis of the response to, and recovery from, sub-lethal cold plasma exposures in P. aeruginosa biofilms improves our current understanding of cold plasma biofilm interactions.

Staphylococcus epidermidis is a major nosocomial pathogen that frequently forms biofilms on indwelling medical devices. This study aimed to investigate the synergistic antimicrobial and antibiofilm activities of octyl gallate (OG) in combination with penicillin and bacitracin against S. epidermidis. Antimicrobial synergy was assessed by conducting checkerboard titration assays, and antibiofilm activity was determined with biofilm assays and fluorescence microscopy analysis. The presence of 8 µg/mL of OG increased both the bacteriostatic and bactericidal activities of penicillin and bacitracin against S. epidermidis. It lowered the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of penicillin by eight-fold and those of bacitracin by four-fold. Moreover, when used with penicillin or bacitracin, OG significantly decreased the level of biofilm production by preventing microcolony formation. Furthermore, OG significantly permeabilized the bacterial cell wall, which may explain its antimicrobial synergy with penicillin and bacitracin. Together, these results demonstrate that OG, a food-grade antioxidant, can be potentially used as a drug potentiator to enhance the antimicrobial and antibiofilm activities of penicillin and bacitracin against S. epidermidis.

Listeria monocytogenes is a foodborne pathogen that can develop serious invasive infections. Among foodborne pathogens, L. monocytogenes exhibits the highest case fatality despite antibiotic treatment, suggesting the current therapy should be improved. Although ampicillin and gentamicin are used as a combination therapy to treat listeriosis, our results showed there is no synergy between the two antibiotics. We discovered that aqueous extract of licorice generated significant antimicrobial synergy when combined with aminoglycosides, such as gentamicin, in L. monocytogenes. In the presence of 1 mg/mL licorice extract, for instance, the minimum inhibitory concentration (MIC) of gentamicin was reduced by 32-fold. Moreover, antimicrobial synergy with licorice extract made gentamicin-resistant clinical isolates of L. monocytogenes susceptible to gentamicin. Given the common use of licorice as a food sweetener in Western countries and a herb in Oriental medicine, our findings suggest that licorice extract can be potentially used as an antibiotic adjuvant to improve the efficacy of antimicrobial treatment of listeriosis.

Dental caries is caused by the buildup of acidic end products that result from the metabolism of dental plaque microbes. Natural products that are widely available could be used as an alternative or adjunctive anti-caries therapy. Sometimes, when two products are used together, they yield a more powerful antimicrobial effect than the anticipated additive effect. These synergistic combinations are often better treatment options because individual agents may not have sufficient antimicrobial action to be effective when used alone. Cranberries contain phenolic compounds like proanthocyanidins (PAC) that disrupt biofilm formation. Manuka honey has high concentrations of the agent methylglyoxal (MGO), which is cariostatic. Because these agents have varied modes of antimicrobial action, they show potential for possible synergistic effects when paired. Various cranberry extracts were tested pairwise with manuka honey or MGO by well-diffusion assays and 96-well checkerboard assays in the presence of Streptococcus mutans to test for synergy. Synergy was demonstrated in cranberry extracts Type R and RE when paired with manuka honey and MGO. The synergistic combinations found in this research thus can be considered candidates for the formulation of a dentifrice that could be used to inhibit the formation of dental plaque and thereby avoid the development of caries. IMPORTANCE The emergence of bacteria resistant to antimicrobial agents has led to a shortage of options when choosing effective treatment agents. Further, some antibiotics used at therapeutic doses can produce undesired side effects. An alternative to traditional antibiotics, natural antimicrobial agents can be used in combination to obtain synergistic outcomes without subjecting the patient to toxic or irritating doses of individual agents. Streptococcus mutans growth and biofilm formation are major contributors to the formation of dental caries. In this study, a synergistic combination of Manuka honey and cranberry extracts gives evidence that it can be used as an alternative or adjunctive anti-caries therapy.

Multidrug-resistant Gram-negative bacteria are a rapidly growing public health threat, and the development of novel antimicrobials has failed to keep pace with their emergence. Synergistic combinations of individually ineffective drugs present a potential solution, yet little is understood about the mechanisms of most such combinations. Here, we show that the combination of colistin (polymyxin E) and minocycline has a high rate of synergy against colistin-resistant and minocycline-intermediate or -resistant strains of Klebsiella pneumoniae. Furthermore, using transcriptome sequencing (RNA-Seq), we characterized the transcriptional profiles of these strains when treated with the drugs individually and in combination. We found a striking similarity between the transcriptional profiles of bacteria treated with the combination of colistin and minocycline at individually subinhibitory concentrations and those of the same isolates treated with minocycline alone. We observed a similar pattern with the combination of polymyxin B nonapeptide (a polymyxin B analogue that lacks intrinsic antimicrobial activity) and minocycline. We also found that genes involved in polymyxin resistance and peptidoglycan biosynthesis showed significant differential gene expression in the different treatment conditions, suggesting possible mechanisms for the antibacterial activity observed in the combination. These findings suggest that the synergistic activity of this combination against bacteria resistant to each drug alone involves sublethal outer membrane disruption by colistin, which permits increased intracellular accumulation of minocycline.

In order to develop bacteriocins, like the lantibiotic nisin A, into effective alternatives to existing antibiotics, their biophysical and physicochemical properties must first be assessed, from solubility, to susceptibility and absorption. It has been well established that formulation strategies at early drug development stages can be crucial for successful outcomes during preclinical and clinical phases of development, particularly for molecules with challenging physicochemical properties. This work elucidates the physicochemical challenges of nisin A in terms of its susceptibility to digestive enzymes like pepsin, pancreatin and proteinase K and its poor solubility at physiological pHs. Low solution concentrations, below the minimum inhibitory concentration against Staphylococcus aureus, were obtained in phosphate buffered saline (PBS, pH 7.4) and in fasted state simulated intestinal fluid (FaSSIF, pH 6.5), while higher solubilities at more acidic pH\'s such as in a KCl/HCl buffer (pH 2) and in fasted state simulated gastric fluid (FaSSGF, pH 1.6) are observed. Tween® 80 (0.01% v/v) significantly increased the solution concentration of nisin A in PBS (pH 7.4, 24 hr). Pancreatin doubled nisin A\'s solution concentration at pH 7.4 (PBS) but reduced its\' inhibitory activity to ∼ 20%, and pepsin almost completely degraded nisin (after 24 hr), but retained activity at biologically relevant exposure times (∼15 min). Harnessing synergism between nisin A and either glycol chitosan or ε-poly lysine, combined with the solubilizing effect of Tween®, increased the antimicrobial activity of nisin A six fold in an in vitro oral administration model.